Pluripotent stem cells, such as human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC), possess the ability to provide an origin for all cell types which are derivatives of the mesoderm, ectoderm and endoderm germinal layers.
In vitro differentiation of pluripotent stem cells to hepatocyte-like cells can potentially generate limitless numbers of cells (Lemaigre, F. P., Gastroenterology, 2009. 137(1), 62-79) with potential for research and therapeutic applications in drug development, detection of drug-induced toxicity, and regenerative medicine. However, the process of differentiation to a specific cell type is often inefficient and lacking in reproducibility. In many instances the competence of cells to acquire an early identity (e.g. definitive endoderm) does not lead to cells being able to successfully commit further to a certain cell type, even when the necessary growth factors and/or small molecules are added in a stage-specific manner (Ochiya, T., Y. Yamamoto, and A. Banas., Differentiation. 2010. 79(2), 65-73).
Definitive endoderm (DE) is formed at approximately 15 days of human embryogenesis and as it gives rise to a variety of organs including liver, its efficient in vitro differentiation is of significant importance. The key finding (D'Amour, K. A., et al., Nat Biotechnol. 2005. 23(12), 1534-41) that exposure of hESC to 100 ng/ml of Activin A in the presence of a low concentration of serum primed a high number of cells to acquire DE identity paved the way for further improvements in differentiation to DE. Activin A was used to mimic Nodal signalling which is crucial during DE development in vivo. While numerous factors have been added to DE specification medium in attempts to improve differentiation (e.g. sodium butyrate, B27 (Hay, D. C., et al., Stem Cells, 2008. 26(4), 894-902; Fletcher, J., et al., Cloning Stem Cells, 2008. 10(3), 331-9); Albumin fraction V, (Cai, J., et al., Hepatology, 2007. 45(5), 1229-39); FGF4 and BMP2 (Hannan, N. R., et al., Nat Protoc, 2013. 8(2), 430-7); Wnt3a and HGF (Chen, Y. F., et al., Stem Cells Dev, 2010. 19(7), 961-78), the use of 100 ng/ml of Activin A as a principal differentiation agent is well established.
Despite these investigations the expression of the pluripotency transcription factors OCT4 and NANOG remain difficult to down regulate effectively (Hay, D. C., et al., Stem Cells, 2008. 26(4): 894-902; Synnergren, J., et al., Stem Cells Dev, 2010. 19(7), 961-78; Touboul, T., et al., Hepatology, 2010. 51(5), 1754-65) suggesting that hESC responses to differentiating factors may be hindered to some extent. There therefore remains a need for simple, cost effective and efficient methods of directing the differentiation process of hESC, iPSC and other primate pluripotent stem cells (pPSC) into cells of the endoderm, mesoderm or ectoderm lineage. In particular, there is a need for a simple and robust method to optimise the differentiation of pluripotent human stem cells to definitive endoderm.
The present invention addresses these problems and provides methods for producing cells of the endoderm, mesoderm and endoderm lineage which have utility in in vitro screening (e.g. for drug development and toxicology studies) and therapy.